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Original Research Article

Medicinal , : Distribution, Ecology, Commercial Value and Use in North Vietnam

Trinh Ngoc Bon1, Trieu Thai Hung1, Phung Dinh Trung1, Tran Cao Nguyen1, Dang Thi Hai

Ha1, Nguyen Thi Hoai Anh1, Hoang Thanh Son1, Tran Hai Long1, Pham Quang Tuyen1, Ninh

Viet Khuong1, Tran Hoang Quy1, Vu Van Nam2 and Tran Van Do1,*

1Silviculture Research Institute, Vietnamese Academy of Forest Sciences, Hanoi, Vietnam

2Lai Chau Forest Protection Department, Dong Phong ward, Lai Chau city, Lai Chau province.

Authors’ contributions

This work was carried out in collaboration among all authors. Author TVD managed the literature searches, performed the statistical analysis, and wrote the first draft of the manuscript. Author PDT performed the statistical analysis. Author TNB, TTH, TCN, DTHH, NTHA, HTS, THL, PQT, NVK, THQ and VVN designed the study and conducted field works. All authors read and approved the final manuscript.

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ABSTRACT

Background:Anoectochilusorchids have been widely used as medicine. However, it has been overharvested in nature because of high commercial values. This study aims at identifying distribution ranges and ecological characteristics of Anoectochilus orchids serving for sustainable development and describing traditional uses by ethnic people in Lai Chau Province, North Vietnam.

Methods: Twenty-one plots of 100 m2 each (10 m × 10 m) were established for ecological survey and stand structures were analyzed for vegetation characteristics. While interviewing was applied to gather information on harvesting method, marketing, and medicinal uses.

Results: Three Anoectochilus orchids(A. lanceolatus, A.calcareus, and A. setaceus)were found in Lai Chau Province, North Vietnam. These species distribute in both evergreen old- growth and secondary broadleaved forests, and mixed broadleaf-bamboo forest on the elevations of 996–2,134 m. They grow in forests with a high canopy cover of 67 m2 canopy area/ 100 m2 land and high cover (>90% land ) of low vegetation (< 2 m tall). It is believed that using the whole plant of Anoectochilus can treat hypertension and diabetes mellitus, and improve the development of underdeveloped children. The folk uses include soaking in alcohol and boiled water for daily drinks like green tea, and consuming as a vegetable. The best harvesting season is during Autumn–Winter and fresh product is mainly marketed with the price of up to 100 U$D/1 kg fresh mass.

Conclusion:A. lanceolatus, A.calcareus, and A. setaceus are potential forest herbs for poverty reduction to ethnic communities in North Vietnam. The natural populations have been reduced remarkably because of overharvesting. Growing techniques are an urgent need for the conservation and sustainable development of such valuable orchids.

Keywords:Climate condition; elevation zone; folk medicine; herb; poverty reduction.

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1. INTRODUCTION

Anoectochilus is a belonging to the family . There are about 50 speciesdescribed in genus Anoectochilus. These species are known asterrestrial,perennial, , sympodial herbs with a creeping , an upright flowering stem, and dark-colored leaves with contrasting veins [1,2]. The flowers are relatively large, hairy and velvety, and arranged in a short spike. While the fruit is a hairy containing winged seeds [3,4].The Anoectochilusorchids have natural distributions from the Himalaya to South , Southeast Asia, , New Guinea, Melanesia, and Hawaii[5]. It is found in evergreen forests with high moisture anddeep shade [1,2]. For example, Anoectochilus roxburghii is found in India, Thailand, China, Laos and Vietnam. Its current status and medicinal uses were studied in Darjeeling Himalaya of West Bengal, India [6], indicating threatened status and value in controlling fever, lung diseases, and hypertension. While indiscriminate collection, deforestation, and extension of agricultural lands are major threats.

Plants of Anoectochilus genus are known as medicinal orchids and have various functions in traditional medicines such as anti-tumor, lipase decreasing, anti-diabetes, and antihepatitis [7]. The chemical constituents and biological activities of some Anoectochilus herbs were described [8-11] such as helvolic acid from A. setaceus for antibacterial activity [12]. The major chemical components in A. setaceusinclude α-cadinol, (E,E)-farnesol, and terpinen-4- ol [13]. While A. formosanus contains β-sitosterol, 4-hydroxycinnamic acid, β-D- glucopyranosyloxy, and butanoidglucosides acid [14]. A. formosanus is used to treat many diseases such as chest pain, stomachache, diabetes, nephritis, high fever, high blood pressure, importance, liver dysfunction, spleen dysfunction, and chest throbbing pain [15]. A. setaceusis used for medicinal oils, treating snakebite poisoning [16,17], and A. roxburghii is used to treat tuberculosis [18]. In addition, Anoectochilus leaves are used as vegetables by local communities and these plantsare also widely used for ornamentations because of their beautiful leaves and flowers [19].

Anoectochilus orchids have been recorded to distribute in high mountainous areas in the Northern provinces of Vietnam [20,21]. It has been widely harvested in nature for commercials and therefore contributed significantly to poverty reduction to ethnic communities. This has led to a remarkable reduction of natural populations. Therefore, understanding the distribution and ecology of Anoectochilus orchids could support the

4 Manuscript was passed through English editing at https://my.enago.com/ development plan and sustainable management for poverty reduction. This work aims at identifying distribution ranges and ecological characteristics of Annoectochilus orchids and describing traditional uses by ethnic people in Lai Chau Province, North Vietnam.

2. METHODS

2.1 Study Site

The study was conducted in Lai Chau Province, North Vietnam (Fig. 1a).Interviewing responsible persons belonging Provincial Department of Forest Development and local citizens was applied to know the potential forests, where Anoectochilus species distribute. The potential areas were first marked on the maps and then assessed in the fields. Old-growth forest (intact and/or less disturbed forests) and secondary forest (forests recovered after shifting cultivation and/or selective logging) were selected for field data collection.

Fig. 1. Map of Vietnam and Lai Chau Province (a), three Anoectochilus species in survey areas (b), and forest cover (c)

2.2. Data Collection

In the field, main plots of 100 m2 each (10 m × 10 m) were established for data collection. All stems with a diameter at breast height (DBH) ≥ 5 cm were identified to species, measured for

DBH (in cm), height (in meter), and crown diameter (Dc in meter). In each main plot, five

5 Manuscript was passed through English editing at https://my.enago.com/ subplots of 4 m2 each (2 m × 2 m) were established in four corners and the middle for a low vegetation survey. All vegetation < 2 m tall were recorded in these five subplots, including species, height, density, and covering (% of land area).

2.3. Commercial Value and Medicinal Use

Interviewing was applied. The interviewees included local ethnic people, Anoectochilus harvesters, and marketers. Interviewing contents included harvesting, marketing, and medicinal uses.

2.4 Data Analysis

Characteristics of stand structure were calculated, including stem density, mean DBH, height, basal area,crown areas, and low vegetation cover. A comparison was conducted by univariate analysis of variance (ANOVA) usingSAS 9.2 at p =0.05.

3. RESULTS

3.1 Distribution and Ecology

Three Anoectochilusorchids were found in the study site, Lai Chau Province, North Vietnam (Table 1), includingAnoectochilus calcareus, A. lanceolatus, and A. setaceus (Fig. 1b). In 21 survey plots, A. calcareus was found in five plots in old-growth broadleaved forests, A. lanceolatuswas foundin four plots in the secondary broadleaved forest, and A. setaceuswas foundin 13 plots in both old-growth and secondary forests of broadleaves and mixed broadleaf-bamboo (Fig. 1c). A. calcareus and A. lanceolatus were grown togetherin one plot, while in other plots only one species was found. The location with the appearances of these three Anoectochilusorchids ranged from 996 m up to 2,134 melevationabove sea level.

All three Anoectochilusorchids were found in evergreen forests of pure broadleaved trees and mixed forest of both broadleaves and bamboo. The forests included both old-growth forest (less disturbed forest) and secondary forest, recovered after shifting cultivation and/or selective logging.The tree stem densities in these study plots ranged 5–12 stems/100 m2, while the range of species number was 3–12. The forests had a mean DBH of 11.6–42.7 cm, height of 6.8–18.0 m. While the largest stem had DBH of 74.5 cm and a height of 26 m. The

6 Manuscript was passed through English editing at https://my.enago.com/ total crown area was in the range of 67.5–343.2 m2/100m2. While in low vegetation of shorter than 2 m tall, the number of plant clumps was in the range of 5–43 clumps/20m2, and land covering of low vegetation was in the range of 90–273.3% (Table 1).

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Table 1. General characteristics of 21 survey plots Low vegetation Plot ELV Canopy layer (per 100 m2 plot; ±SE) (per 20 m2) (m) Anoectochilusorchids Stems Species DBH (cm H Basal area Crown area Clumps DBH (cm) max H (m) Cover (%) ) (m) max (m2) (m2)

1 1,930 10 6 15.5 ±3.2 43.6 8.4 ±0.6 11.0 0.263 139.0 27 156.2 Anoectochilus calcareus 2 2,057 5 4 18.8 ±5.1 29.6 8.9 ±1.7 13.0 0.179 107.0 24 237.8 Anoectochilus calcareus 3 2,123 7 6 25.6 ±6.8 58.3 14.0 ±2.4 24.0 0.513 146.1 5 100.0 Anoectochilus calcareus 4 2,134 6 6 42.7 ±8.7 74.5 18.0 ±1.7 24.0 1.038 175.9 15 141.5 Anoectochilus calcareus 5 1,930 9 8 17.2 ±2.5 27.7 9.7 ±0.7 12.0 0.243 168.9 14 119.0 A. calcareus and A. lanceolatus 6 1,906 5 4 15.5 ±2.0 20.4 8.8 ±1.0 12.0 0.100 67.5 23 99.9 Anoectochilus lanceolatus 7 1,894 7 4 13.4 ±2.3 23.2 8.1 ±1.2 14.0 0.116 97.4 19 131.7 Anoectochilus lanceolatus 8 1,915 8 8 16.2 ±2.2 26.9 10.9 ±1.0 17.0 0.185 134.5 27 198.9 Anoectochilus lanceolatus 9 1,395 12 10 11.6 ±1.3 23.6 8.7 ±0.8 14.0 0.143 134.3 51 184.6 10 1,434 12 9 14.3 ±1.5 24.8 9.3 ±0.7 13.0 0.216 106.8 26 244.0 Anoectochilus setaceus 11 1,009 7 3 19.9 ±2.3 26.1 12.6 ±0.6 14.0 0.234 166.5 38 174.4 Anoectochilus setaceus 12 996 6 5 14.4 ±3.5 26.4 9.8 ±1.6 15.0 0.127 106.0 30 272.5 Anoectochilus setaceus 13 1,010 6 6 18.5 ±3.6 29.0 10.3 ±1.3 14.0 0.191 81.7 37 136.1 Anoectochilus setaceus 14 1,192 8 4 16.6 ±2.4 24.5 9.8 ±0.9 13.0 0.200 125.7 35 209.5 Anoectochilus setaceus 15 1,220 8 6 18.7 ±4.7 39.5 11.2 ±1.8 19.0 0.318 168.1 39 273.3 Anoectochilus setaceus 16 1,208 7 7 14.4 ±3.3 30.9 9.9 ±1.4 16.0 0.149 84.0 35 222.1 Anoectochilus setaceus 17 1,229 9 8 17.0 ±2.8 29.9 11.1 ±1.3 17.0 0.248 118.6 38 219.8 Anoectochilus setaceus 18 1,472 7 7 13.4 ±3.0 24.8 6.8 ±0.6 8.5 0.128 48.7 38 90.0 Anoectochilus setaceus 19 1,472 8 7 11.6 ±1.2 16.2 8.4 ±0.8 12.0 0.090 74.6 35 138.5 Anoectochilus setaceus 20 1,459 7 6 10.1 ±0.9 12.7 8.3 ±0.9 11.0 0.059 63.6 32 126.1 Anoectochilus setaceus 21 1,473 7 7 35.2 ±7.0 65.9 17.0 ±2.1 26.0 0.842 343.2 43 236.7 Anoectochilus setaceus ELV is elevation above sea level

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There were significant differences of structures (distribution elevation, low vegetation, crown area of canopy trees, mean DBH, and size of largest trees) among stands of three orchids (Table 2). A. setaceus prefers to distribute on the lowest elevation of around 1,264 m above sea level, the distribution elevation (1,777 m) is higher in A. lanceolatus, and the highest distribution elevation (2,034 m) was found in A. calcareus. Low vegetation significantly affected the distribution of three orchids as A. setaceus distributes in the highest cover of 195% and numerous vegetation clumps of 35/20 m2, reducing to A. lanceolatus with a cover of 153% and 29 clumps/20 m2. While A. calcareus seems to distribute in the lowest vegetation cover of 150% and vegetation clump of 17/20 m2 (Table 2). In addition, A. calcareus grows in the old-growth forest with the highest difference of stem sizes includingDBH and height. While A. lanceolatus grows in secondary forest with much similar stems sizes.

The differences of stand structures in terms of stem density, height, and maximum height among three orchids were not significantly different (Table 2). Which were in the range of 7– 8 stems/100 m2 and canopy height of 9–11 m.

Table 2. Comparisons of stand structures (±SE) among three Anoectochilus orchids

Canopy layer (per 100 m2 plot) Low vegetation ELV (m) Anoectochilusor (per 20 m2) chids Stems DBH DBHmax(c H Hmax( Crown Clump Cover (cm) m) (m) m) area (m2) (%) 7.4 23.9a 46.7a 11.8 16.8 147.4a 17.0a 150.9a 2,034a A. calcareus ±0.9 ±5.0 ±8.9 ±1.8 ±3.0 ±12.2 ±3.9 ±23.7 ±44.8 8.0 14.2b 23.5b 9.1 14.2 108.4b 29.9ab 153.7a 1,777b A. lanceolatus ±1.5 ±1.0 ±1.3 ±0.6 ±1.0 ±16.2 ±7.2 ±23.1 ±127.6 7.6 17.0c 29.2c 10.3 14.8 123.9ab 35.5b 195.2b 1,264c A. setaceus ±0.5 ±1.9 ±3.9 ±0.7 ±1.3 ±22.6 ±1.3 ±17.6 ±55.7 Different letters a, b, c in a indicate significant difference of means at p =0.05. ELV is the elevation above sea level

Structure of crown area distribution in different height classes of tree stems was different among stands of three orchids (Fig. 2). A. lanceolatus grows in simplest canopy forest, which has only four height classes and less crown area (Fig. 2b) with the highest crown area in the stems of 9–13 m tall. While A. calcareus and A. setaceus grow in forests with more complex canopy, which has five height classes (Fig. 2a, c). Between these two orchids, there are also differences. The highest crown area focused on stems of 14–21 m tall in A. calcareus (Fig.

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2a), while that was in stems of > 17 m tall in A. setaceus, which is double that in stems of < 17 m tall (Fig. 2c).

120 120 120 ) ) ) 2 (a) 2 (b) 2 (c) 90 90 90 /100 /100 m /100 m /100 m 2 2 2 60 60 60

30 30 30 Crown area (m area Crown (m area Crown (m area Crown 0 0 0

<9 9-13 14-1717-21 >21 <9 9-13 14-1717-21 >21 <9 9-13 14-1717-21 >21 Height class (m) Height class (m) Height class (m) Fig. Fig 2. Vertical crown structure for A. calcareusstand (a), A. lanceolatusstand (b), and A. setaceusstand (c). Vertical bars indicate +SE

3.2 Commercial Value and Medicinal Use

The price of Anoectochilus orchids is depending on species. A. lanceolatus costs 0.5 U$D/1 kg fresh mass, it is 50 U$D/1 kg fresh mass for A. calcareus and 100 U$D/1 kg fresh mass for A. setaceus. Generally, after harvesting from natural forests the fresh product is sold at homes or local markets to outside consumers, who will resell to trading companies of China mostly. The best harvesting season is during Autumn-Winter when Anoectochilushas the highest production and quality by traditional knowledge. It was recorded that the availabilityof Anoectochilusin natural forests 10 years ago was numerous, which can be easily found at any old-growth forest. Recently, it is difficult to find out Anoectochiluseven in remote old-growth forests. A person takes one day to harvest 0.2 kg fresh mass of Anoectochilus nowadays, compared to several kilograms in the past ten years. Around 80% harvest product is marketed and the rest is used by local people.

In the study site, the whole plant of Anoectochilus is used to treat hypertension and diabetes mellitus. Underdeveloped children can use this to improve appetite and support digestion. Folk uses include soaking in alcohol for daily drinks and soaking in boiled water like green tea, or consuming as vegetables.

4. DISCUSSION

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4.1 Distribution and Ecology

Anoectochilus species are terrestrial , growing in soil, leaf litter, humus, and also on mossy rocks in lowland and mid-mountain forests up to 2,135 m elevation [6,22]. Like other forest herbs [23,24],Anoectochilus is shade-tolerant plants (Fig. 1 b, c), which can only survive and grow under shade. The shading level is different among Anoectochilus species [25]. This was confirmed in the present study as the differences of stand structures among three Anoectochilusspecies, especially in terms of low vegetation and crown area (Table 2; Fig. 2). In addition, in 20 of 21 plots surveyed only single Anoectochilus orchid was found in a plot. This indicated the difference in ecology requirements among three orchids. Therefore, it is difficult for these orchids to grow in the same vegetation. The many differences of vertical crown structures among three orchids (Fig. 2) indicate the difference of light regime penetrating to the forest floor, which plays an important role in survival and growth offorest herbs.

Anoectochilus lanceolatus Lindl is found in the , Nepal, Bangladesh, Myanmar, Taiwan, Thailand, and Vietnam [5]. It grows on shaded rocks in deep shade broadleaved forests on elevations of 800–2,200 m [26]. In the present study, the species was found in North Vietnam (22o07’43.3’’N and 103o54’44.2’’E) on the elevation of 1,894–1,915 m close to streams with high humidity. The species was found in both secondary and old-growth evergreen broadleaved forest. It grows in deep shade evergreen broadleaved forest but even can grow in secondary forest with some levels of human disturbance, while still keeps suitable canopy cover. The plants were only found near water bodies, growing on soil with a high humus layerand high humidity, indicating the importance of humidity on the distribution and growth ofA. lanceolatus.

Anoectochilus calcareus Averis native species of Vietnam andfound in evergreen coniferous and broadleaved forests on rocky limestone in northern Vietnam on the elevations of 450– 1,600 m [27]. In the present study, the species was found in North Vietnam (22o08’20.1’’N and 103o54’34.3’’E) on the elevation of 1,930–2,134 m in the old-growth evergreen broadleaved forest. The elevation range is higher than previously reported, indicating a wider distribution range of A. calcareus. In addition, it was found growing on soil other than rocky limestone previously reported [27]. This again indicates the wider ecological conditions for distribution and growth of A. calcareus, and therefore it could be found to have natural distributions further to southern Vietnam.

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Anoectochilus setaceus Blumeis found in China, India, Bangladesh, Nepal, Himalayas, Sri Lanka, Myanmar, Thailand, Vietnam, and Indonesia. The species grows in evergreen broadleaved forests on the elevations of 1,200–1,800 m [28,29]. In the present study, it was found in North Vietnam (22o44’35.4’’N and 103o24’56.7’’E) on the elevation of 996–1,473 m in both old-growth and secondary evergreen broadleaved forests and mixed broadleaf- bamboo forest in all foot, middle and top of the mountains. The lower elevation distribution found in the present study indicates the wider distribution of A. setaceus. In addition, it can grow in several types of vegetation which have high shading levels and in all mountain positions. Therefore, ecological conditions for A. setaceus are quite wide. But the most important condition is the vegetation cover. It should be high enough to ensure shading for this shade-tolerant orchid (Table 2; Fig. 2c).

The natural populations of Anoectochilus are under threat from overharvest, habitat reduction, and forest fragmentation [6,7,19,30]. Therefore, in each region and country where such plants are now available[6], preservation plan should be established for successful protection and future development. The commercial price of these three Anoectochilus orchids is much different. This is believed to relate to the medicinal values of compounds in each species. Therefore, further studies on medicinal compounds must be conducted for further recommendations on conservation, usage, and development.

4.2 Commercial Value and Medicinal Use

Anoectochilus species have been widely used as folk medicine in China, Sri Lanka, Taiwan, and Vietnam for centuries [22,31,32].It is used to treat cancer, hypertension, diabetes mellitus, lung and liver diseases, nephritis, impotence, spleen disorder, pleurodynia, and underdeveloped children [33-35]. Therefore, currently, it has been widely used in modern medicines. For example, quercetin-7-O-β-D -[6”-O-(trans-feruloyl)]-glucopyranoside compound isolated from an extract of A. roxburghiiexhibits strong scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl free radical [36]. Anoectochilus orchids could be considered as potential plants for both folk and modern medicines recently and in the future.

With a high price of up to 100 U$D/1 kg fresh mass of A. setaceus in Vietnam recently, it is a potential forest herb for poverty reduction. As the local community in mountainous areas is living in a much lower standard compared to other parts of Vietnam. However, growing

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Anoectochilus is not easy work and the plants grow quite slow. Growing Anoectochilus orchids in local communities for production purposes is not available other than for ornamentation. Therefore, researching for simple and applicable growing techniques for Anoectochilus orchids should be conducted and the best one must be recommended for practical application.

5. CONCLUSION

Anoectochilus lanceolatus, A.calcareus, and A. setaceus are three orchids found in Lai Chau Province, North Vietnam. These species distribute in evergreen old-growth and secondary broadleaved forests, and mixed broadleaf-bamboo forest on the elevations of 996–2,134 m above sea level. These orchids are used locally by soaking in alcohol and boiled water for daily drinks like green tea, and consuming as vegetables. This is believed to have positive impacts on hypertension, diabetes mellitus, and underdeveloped children. Such high medical values have led to remarkably reduced populations of Anoectochilus orchids in nature. Therefore, researches on growing, harvesting, and processing are an urgent need, which can contribute to preserving and developing Anoectochilus orchidsin North Vietnam sustainably.

CONSENT

It is not applicable.

ETHICAL APPROVAL

It is not applicable.

ACKNOWLEDGEMENT

This research is funded by the Lai Chau Department of Science and Technology. We would like to thank anonymous reviewers for constructive comments on the manuscript.

COMPETING INTERESTS

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Authors have declared that no competing interests exist.

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